1. Field of the Invention
This invention relates to the manufacture and use of recombinant albumin fusion proteins to make human interferon analogs. The novel interferon analogs have the same functions with interferon in bio-assays, in vitro or in vivo. These long acting recombinant interferon analogs that are particularly expressed in yeast can largely improve interferon's therapeutic function.
2. Description of Related Art
1. Albumin
Albumin is a soluble, monomeric protein that comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilizing extracellular fluid volume. Mutations in this gene on chromosome 4 result in various anomalous proteins. Albumin is a globular un-glycosylated serum protein of molecular weight 65,000. The human albumin gene is 16,961 nucleotides long from the putative ‘cap’ site to the first poly(A) addition site. It splits into 15 exons which are symmetrically placed within the 3 domains that are thought to have arisen by triplication of a single primordial domain. Albumin is synthesized in the liver as pre-pro-albumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin. HSA has 35 cysteins; in blood this protein monomer has 17-disulfide linkage (Brown, J. R. “Albumin structure, Function, and Uses” Pergamon, N.Y., 1977). HSA is misfolded when produced intracellularly in yeast without its amino terminal secretion peptide sequence. This conclusion is based on its insolubility, loss of great than 90% of its antigenicity (as compared to human-derived HSA), and formation of large protein aggregates. At present albumin for clinical use is produced by extraction from human blood. The production of recombinant albumin in microorganisms has been disclosed in EP 330 451 and EP 361 991.
Albumin is a stable plasma transporter function provided by any albumin variant and in particular by human albumin. HSA is highly polymorphic and more than 30 different genetic alleles have been reported (Weikamp L, R, et al., Ann. Hum. Genet., 37 219-226, 1973). The albumin molecule, whose three-dimensional structure has been characterized by X-ray diffraction (Carter D. C. et al., Science 244, 1195-1198, 1989), was chosen to provide the stable transporter function because it is the most abundant plasma protein (40 g per liter in human), it has a high plasma half-life (14-20 days in human, Waldmann T. A., in “Albumin Structure, Function and Uses”, Rosenoer V. M. et al (eds), Pergamon Press, Oxford, 255-275, 1977), and above all it has the advantage of being devoid of enzymatic function, thus permitting its therapeutic utilization at high dose.
2. Interferons
Interferons are a heterogeneous family of multifunctional cytokines whose first demonstrated biological activity was the induction of cellular resistance to virus infection. Antiviral activity of interferon was the only recognized biological function of the interferons for many years. Today interferons are found many other bio-functions. Interferon's actions on cell growth and differentiation and their many immunoregulatory activities are probably of greater fundamental biological significance.
Two very distinct families of proteins are counted among the interferons. The IFN-α/β “superfamily” (also called type I IFN) encompasses a group of structurally related genes and proteins that are further subdivided into the subfamilies IFN-αI IFN-αII, and IFN-β. The second “family” consists of a single gene encoding a single protein termed IFN-γ (also called type II IFN or immune IFN). It should be made clear at the outset that IFN-γ is structurally unrelated to the members of the IFN-α/β superfamily. The reasons for discussing IFN-α/β and IFN-γ together are largely historical. Interferon was first described by Isaacs and Lindenmann (1957) as a product of virus-infected cells capable of inducing resistance to infection with homologous or heterologous viruses. A functionally related virus inhibitory protein (today termed IFN-γ) was described by Wheelock (1965) as an “Interferon-like” substance produced by mitogen-activated T-lymphocytes. For many years the only properties that made it possible to distinguish IFN-γ from the other interferons were its lack of stability at Ph 2 (Wheelcok 1965) and distinct antigenic specificity (Youngner and Salvin 1973). Only when the sequences of the proteins and genes of the major interferons were revealed in the early 1980s did it become clear what the relationship of the different interferons is to each other. People recognize now that IFN-γ is primarily an immunoregulatory cytokine whereas the potential actions of IFN-α/β extend to a broader variety of cells and tissues.
Members of the IFN-α/β superfamily represent the classical interferons. The first clear indication of the heterogeneity of the type I interferon proteins came from studies showing that interferons derived from human leukocytes and fibroblasts are antigenically distinct (Havell et al. 1975). Eventually leukocyte and fibroblast interferons were designated IFN-α and -β, respectively (COMMITTEE ON INTERFERON NOMENCLATURE 1980). Most of the information on interferon structure has been derived from gene cloning studies. At least 24 nonallelic human IFN-α genes and pseudogenes have been identified. They can be divided into two distinct subfamilies, termed IFN-αI and -αII (Weissmann and Weber 1986). The IFN-αI subfamily potentially functional genes and several pseudogenes. The IFN-αII subfamily is known to comprise only one functional gene and five or six nonallelic pseudogenes. IFN-αI genes encode mature proteins consisting of 165-166 amino acids; IFN-αII gene encodes a mature protein 172 amino acids long. All of the genes encode N-terminal secretive signal peptide presequences (generally 23 residues long) which are removed by proteolytic cleavage before the release of the mature interferon molecule from the cell. While it is clear that a high degree of homology is found among all human IFN-α genes and proteins, the IFN-αII sequences have diverged significantly from the -αI sequences, warranting their classification into a separate subfamily (Capon et al. 1985). In fact, it has been suggested that the IFN-αII subfamily be named IFN-ω (Adolf 1987).
IFN-α forms vary in molecular mass between 19 and 26 kDa and are produced by monocytes/macrophages, lymphoblastoid cells, fibroblasts, and a number of different cell types following induction by viruses, nucleic acids, glucocorticoid hormones, and low-molecular weight substances. The effects of IFN-α are wide ranging and include potent anti-viral and anti-parasitic activity. In addition, IFN-α has anti-proliferative effects on certain tumor cells. Human IFN-α species lack potential N-glycosylation sites and most members of the IFN-α subfamilies in their native state are not glycosylated (Pestka 1983). Several natural human IFN-α proteins have been purified to homogeneity. They were shown to range in their apparent molecular weights from 16000 to 21000 (Rubinstein et al. 1981). The reason for these large differences in the apparent molecular weights has not been fully explained.
A single gene for human IFN-β encodes a 166-residue-long mature protein. Homology between IFN-β and members of the IFN-αI subfamily is about 25-30% at the amino acid level and about 45% in the coding sequences at the nucleotide level (Taniguchi et al. 1980). In addition, there is also extensive homology in the 5′nucleotide flanking regions which contain transcriptional promoter and enhancer sequences, reflecting the fact that IFN-α and -β genes are often coordinately induced (Degrave et al. 1981).
Interferons represent an important class of biopharmaceutical products, which have a proven track record in the treatment of a variety of medical conditions, including the treatment of certain autoimmune diseases, the treatment of particular cancers, and the enhancement of the immune response against infectious agents. To date, five types of interferons have been found in humans: interferon-alpha, interferon-beta, interferon-gamma, interferon-omega and a new form of human and murine interferon, “interferon-.epsilon.,” which have applications in diagnosis and therapy.
Interferon is used for treatment of Hepatitis C, B, and broad range of cancers, such as chronic myelogenous leukemia. Hepatitis C is an inflammation of the liver caused by hepatitis C virus infection. The HCV is most common chronic blood-borne disease in China (almost 80 millions HCV carrier) and USA (almost 4 millions HCV carriers), which causes 1 million people death worldwide per year. Chronic hepatitis B is an inflammation of the liver caused by HBV. The HBV infection can be developed into liver cancer and cirrhosis. 500 million people are infected by HBV in worldwide.
Production of IFN-α/β during virus infections is generally beneficial as it serves to limit the spread of virus and promote recovery (Gresser et al. 1976). In the past few years several types of interferon preparations have been licensed for clinical use. In the United States E. coli-derived recombinant human IFN-α 2 (IFN-α-2a) and IFN-α A (IFN-α-2b) have been approved for use in the treatment of hairy cell leukemia. IFN-α 2 and IFN-α A are both members of the IFN-αI subfamily and they differ from each other in a single amino acid in position 23 (Arg in α2 and Lys in α A). One of the preparations has also been approved for the treatment of condylomata acuminata. Other interferon preparations also have been approved for clinical use in some countries, e.g., a natural mixture of several IFN-α subtypes produced in the Namalwa line of human lymphoblastoid cells or natural human IFN-β produced in cultured fibroblasts. The approved use of these interferon preparations some countries includes chronic active hepatitis B, acute viral encephalitides, and nasopharyngeal carcinoma. A preparation of E. Coli-derived recombinant human IFN-γ has been approved for therapeutic use in rheumatoid arthritis in the German Federal Republic. Approved and experimental therapeutic applications of interferons have been extensively covered in a volume devoted to this topic (Finter and Oldham 1985). Interferon-beta, preferably in low doses, is used for stimulation of erythropoiesis in disorders characterized by lack of maturation of progenitor blood cells to red cells, (Michalevicz, U.S. Pat. No. 5,104,653)
Novel polypeptide produced by E. coli transformed with a newly isolated and characterized human IFN-.alpha and the gene is described. The polypeptide exhibits interferon activities such as antiviral activity, cell growth regulation, and regulation of production of cell-produced substances. Those novel interferon are named as Interferon-α-67, by Innis, in U.S. Pat. No. 5,098,703; Interferon-.alpha.54, in U.S. Pat. No. 4,975,276, and Interferon-.alpha.61, in U.S. Pat. No. 4,973,479.
Therapeutically synergistic mixtures of purified gamma interferon and purified interleukin-2 are provided for treatment of tumor-bearing hosts. Preferably, the gamma interferon and interleukin-2 are obtained from recombinant cell synthesis (Palladino U.S. Pat. No. 5,082,658).
The invention provides fusion proteins comprising an N-terminal region derived from an interferon-tau (IFN-.tau.) polypeptide and a C-terminal region derived from another type I interferon polypeptide, such as IFN-.alpha. or IFN-.beta. The fusion proteins exhibit reduced cytotoxicity compared to the corresponding unmodified type I interferons. Johnson, et al. U.S. Pat. No. 6,174,996 is the only patent that mentions how to make an interferon fusion protein.
A method that comprises administering a PEG.sub.12000-IFN alpha conjugate to an individual afflicted with a viral infection susceptible of treatment with interferon alpha, preferably chronic hepatitis C, is disclosed. Glue et al. U.S. Pat. No. 5,908,621 is a patent mentions how to make a long acting or slow release form interferons. Shechter et al., (Proc. Natl. Acad. Sci. USA. 2001 Jan. 30; 98 (3): 1212-1217) reported the method to prolong the half-life of human interferon-α2 in circulation by covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-α2.
There is an invention that features a novel hybrid interferon species that comprises a chain of 161 and/or 162 amino acids. The hybrid is novel not only because its new structure, but also for the reason that the hybrid comprises a shortened or truncated segment of alpha interferon. Hence, an entirely new interferon species which does not occur in nature is reported by Leibowitz et al. in U.S. Pat. No. 4,892,743.
Chang et al. in U.S. Pat. No. 5,723,125 patent disclosed a hybrid recombinant protein consisting of human interferon, preferably interferon-.alpha. (IFN.alpha.), and human immunoglobulin Fc fragment, preferably .gamma.4 chain. These two protein fragments are joined by a peptide linker comprising the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser. This method makes an interferon-α fusion protein.
Kriegler, et al. in U.S. Pat. No. 5,324,655 patent reported a virion expression system for a desired protein packaged in an envelope derived from a retrovirus useful in administering proteins which cross cell membranes in order to serve their function. Preferred virions are those that carry an RNA sequence that encodes cytokines or lymphokines, and includes IL-2, multiple drug resistance protein, and TNF. Particularly disclosed is a DNA construct in which a gene encoding tumor necrosis factor (TNF) is directly linked to DNA encoding a human gamma-interferon signal peptide.
There are some research paper reported that the combination use of interferons could bring some beneficial to patients such as Trotta in U.S. Pat. No. 5,190,751 patent reported the human leukemia T-cells and B-cells are inhibited from proliferating by treatment with a combination of recombinant human alpha and gamma interferons, either simultaneously or sequentially, and the alpha interferon is preferably recombinant human alfa-2b interferon.
A common feature for any of these administration modes, however, is rapid inactivation of IFN-α in body fluids and in various tissues (O'Kelly, et al., 1985. Proc. Soc. Exp. Biol. Med. 178, 407-411). This in turn leads to the disappearance of the cytokine from the plasma within several hours after administration (Rostaing, et al., 1998, J. Am. Soc. Nephrol. 9, 2344-2348). Unlike many other administered protein drugs, the major route of IFN-α elimination in vivo takes place in the circulatory system through proteolysis and inactivation by serum proteases. Therefore, long acting of interferon is needed in treatment of patients with viral infection or cancers in clinical trials.